A Thermally regulated, Oligonucleotide-mediated one-Pot System for CRISPR-Cas12a (TOPS-CRISPR), which employs a programmable inhibition strategy based on complementary RNA blockers with tunable length and binding sites, enabling efficient and reversible steric inhibition of the LbCas12a-crRNA ribonucleoprotein (RNP) complex, resolving the inherent contradiction between amplification and cleavage in one-pot assay.
Abstract
CRISPR-Cas12a has emerged as a powerful tool in molecular diagnostics, owing to its robust signal amplification and compact crRNA design. However, its uncontrolled enzymatic activity often hampers application in streamlined one-pot assays. Although existing temporal or spatial regulation strategies can mitigate this issue, they typically introduce operational complexity or increased cost. Here, we designed a Thermally regulated, Oligonucleotide-mediated one-Pot System for CRISPR-Cas12a (TOPS-CRISPR), which employs a programmable inhibition strategy based on complementary RNA blockers with tunable length and binding sites, enabling efficient and reversible steric inhibition of the LbCas12a-crRNA ribonucleoprotein (RNP) complex, resolving the inherent contradiction between amplification and cleavage in one-pot assay. TOPS-CRISPR not only is operational simple and cost-effective but also achieves over 60-fold higher sensitivity than conventional one-pot platforms. We demonstrated the clinical applicability of TOPS-CRISPR by accurately detecting Brucella and Streptococcus in both spiked and clinical samples. Moreover, the system integrates seamlessly with rapid sample processing, lyophilized reagents, and miniaturized workflows, enabling field-deployable pathogen identification within 50 min.
The CRISPR/Cas12a system has revolutionized molecular diagnostics due to its RNA-guided trans-cleavage activity, enabling programmable and highly accurate nucleic acid detection. However, most Cas12a-based assays are optimized for DNA targets, while direct RNA detection constrained by limited sensitivity, typically at the nanomolar level. Existing strategies to improve the performance of RNA analysis often rely on additional DNA activators or complex auxiliary systems. Here, we report a simple yet effective chemical additive-based strategy that overcomes these limitations. This chemical additives-enhanced CRISPR/Cas12a-based RNA detection (CARD) enables femtomolar-level RNA detection using only a single crRNA, without the need for DNA activators, reverse transcription, or strand-displacement reactions. Notably, this approach can be adapted to single-stranded DNA, enabling ssDNA detection at attomolar levels. Collectively, CARD provides a straightforward, amplification-free, and highly sensitive diagnostic framework that might be readily extended to other CRISPR/Cas systems for ultrasensitive nucleic acid diagnostics.
Jun Chen, Haiyan Zheng, Lucas Guan et al.· Biosensors & bioelectronics· 0 citations
Abstract Selective and tunable regulation of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a activity enables on-demand control, yet current strategies remain hindered by nonspecific regulation and limited tunability. Inspired by proximity effect, we present a Cas-regulation-targeting chimera (CasTAC) strategy that employs CRISPR RNA (crRNA) as a proximity mediator to carry phosphorothioate regulators to interfere with catalytic or recognition domains of Cas12a and consequently suppress its activity. This crRNA-induced proximity approach can effectively eliminate nonspecific interaction between phosphorothioate regulators and proteins within complex multi-enzyme systems, thereby enabling selective control over CRISPR/Cas12a activity. Furthermore, CRISPR/Cas12a activity can be finely tuned to different inhibitory levels by varying the number of phosphorothioate regulators. The CasTAC strategy also improves nuclease resistance and single-nucleotide discrimination, offering potential advances in the sensitivity of molecular diagnostics and the accuracy of gene editing. Notably, the CasTAC balances the kinetics of nucleic acid amplification and CRISPR cleavage, facilitating efficient product accumulation and resolving compatibility issues in one-pot assays. As a proof of concept, we develop a one-pot, one-step recombinase polymerase amplification–CasTAC assay that achieves over 1000-fold higher detection sensitivity than the conventional one-pot recombinase polymerase amplification−CRISPR/Cas12a assay. The CasTAC strategy provides a versatile framework for fine–tuning Cas activity and advances CRISPR technology toward refined and context-adaptable functionality.
CRISPR/Cas12a holds great promise for biosensing and diagnostics, but conventional methods suffer from low catalytic efficiency, high background, and reliance on pre-amplification. Direct detection of structured RNAs also remains challenging. Herein, we report the development of a reverse transcriptase and LNA probe (LNA-p)-mediated CRISPR/Cas12a positive feedback system (RTLC) for highly efficient, one-pot detection of both DNA and RNA. Without pre-amplification or thermal cycling, the assay achieves a 0.5 aM detection sensitivity within 27 min, exhibits single-base resolution, and allows direct detection of RNAs up to 985 nt in length. Together, RTLC is successfully validated in practical samples by detecting lncRNA HULC and miR-21, offering a robust, versatile tool for high-performance nucleic acid diagnostics.
The potential of terminally engineered PAM-less dsDNA as a structural handle for programming Cas12a activity is highlighted and useful insight is provided for the design of CRISPR-based biosensing strategies.
A one-pot self-primer isothermal exponential amplification reaction (SP-EXPAR) combined with a CRISPR/Cas14a assay was developed for detecting KRAS G12C and G12D and demonstrated 100% sensitivity and 100% specificity compared with DNA sequencing.
Guozhi Yang, Yaqin Chen, Wenyong Zhao et al.· Analytical Methods· 0 citations
Molecular diagnostic technologies play an indispensable role in modern medicine and public health. However, traditional diagnostic platforms frequently face an inherent trade-off between laboratory-grade analytical precision and the speed and operational simplicity required for point-of-care testing. In recent years, the emergence of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) protein system has precipitated disruptive technological changes to this field. CRISPR-Cas system possesses high-fidelity target recognition capability and exhibits a distinctive trans-cleavage activity upon activation, which functions as signal amplification. This technology alleviates the inherent trade-off between sensitivity and portability. This review systematically summarizes the core molecular mechanisms of CRISPR-Cas detection platforms, addressing the differences in substrate preference and cleavage behavior among mainstream effector proteins (e.g., Cas9, Cas12, Cas13, and Cas14) and prokaryotic Argonaute (pAgo) proteins. Furthermore,this review sorts out the technological iteration path of detection platforms and presents the applications of this technology in fields such as infectious disease surveillance, cancer liquid biopsy, preliminary screening of genetic diseases, food and environmental safety, and veterinary port quarantine. Despite the challenges in quantitative accuracy and anti-interference ability, CRISPR biosensors are powerfully driving precision medicine towards decentralized, on-site, and accessible Point-of-Care Testing (POCT).
